Cell Cycle Regulation by Carboxylated Multiwalled Carbon

Article pubs.acs.org/crt

Cell Cycle Regulation by Carboxylated Multiwalled Carbon Nanotubes through p53-Independent Induction of p21 under the Control of the BMP Signaling Pathway Yi Zhang†,§ and Bing Yan‡,§,* †

School of Pharmaceutical Sciences and ‡School of Chemistry and Chemical Engineering, Shandong University, Jinan 250100, China Department of Chemical Biology and Therapeutics, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105, United States

§

S Supporting Information *

ABSTRACT: This report describes how carboxylated multiwalled carbon nanotubes (MWCNTs) induce p53-independent p21 expression and cell cycle arrest. MWCNTs suppress BMP signaling and lead to the downregulation of Id protein production and the upregulation of p21 because p21 expression is directly controlled by Id proteins through their regulation of the E-box motifs in the p21 promoter. The overexpressed p21 protein then binds to the cyclin D/cdk4,6 complexes and inhibits the phosphorylation of Rb protein. Hypophosphorylation of Rb prevents the release of E2F factors and causes cell cycle arrest. These findings provide valuable insight into a mechanistic understanding of carbon nanotubes’ effects on cellular functions.

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aneuploidy in primary human respiratory epithelial cells19 and induce malignant cell transformation of human lung epithelial cells.20 Both events are linked to cell cycle alterations or the loss of cell cycle checkpoint control.21,22 These observations imply that CNTs may alter cell cycle regulation. Although CNTinduced cell cycle arrest was reported,23−25 the likely perturbation of CNTs on cell cycle checkpoints is not well known. Carboxylated MWCNTs, a more soluble form of CNTs, may have greater biomedical and other applications. A full understanding of their biological activities is needed to broaden their applications and circumvent their latent toxicity. In this study, we found that MWCNTs altered the cell cycle without causing apoptosis in a panel of cell lines by inducing the expression of the p21 protein. The overexpression of p21 is p53-independent and is a result of MWCNTs’ suppression of the BMP signaling pathway, which leads to the downregulation of Id proteins. The reduced expression of Id proteins alters the control of the E-box motifs of the p21 promoter that upregulates p21 expression. The p21 binds to the cyclin D/ cdk4,6 complex and reduces Rb phosphorylation that leads to cell cycle arrest.

INTRODUCTION Nanotechnology has impacted most industrial sectors such as energy,1 materials,2 environment,3 and information technology.4 As of today, there are more than 1310 nanotechnologybased consumer products on the market according to the Woodrow Wilson International Center for Scholars.5 Among them, at least 31 are based on CNT materials. CNTs have also been extensively explored for their applications in biomedicine,6 including drug delivery,7,8 tumor imaging,8−10 and tissue engineering.11 These exploding applications of CNTs have raised concerns over their environmental and human health risks. However, the biological consequences of human exposure to CNTs and the associated molecular mechanisms are still to be elucidated. Preliminary investigations have shown that CNTs induce inflammation, epithelioid granulomas, and interstitial or pleural fibrosis in mouse models.12 Although the molecular mechanisms by which CNTs cause toxicity are not well-known, reactive oxygen species (ROS) has been proposed as one of the possible causes.13 Eukaryotic cell cycle progression is under the control of a network of regulatory pathways named cell cycle checkpoints.14 Cell cycle progression is arrested when cells (i) repair cellular damage; (ii) dissipate an exogenous cellular stress signal; or (iii) need essential growth factors, hormones, or nutrients.15 Checkpoint signaling may also lead to programmed cell death if cellular damage overwhelms the repair capability.16 The dysregulation of cell cycle checkpoints can lead to various diseases17 or tumorigenesis.18 CNTs were reported to cause © 2012 American Chemical Society

Received: February 13, 2012 Published: March 19, 2012 1212

dx.doi.org/10.1021/tx300059m | Chem. Res. Toxicol. 2012, 25, 1212−1221

Chemical Research in Toxicology

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cycle consisting of denaturation at 94 °C for 30 s, annealing at 62 °C for 30 s, and extension at 72 °C for 1 min. The primers used are as follows: p21, forward, 5′-ACAGTTGGTCAGGGACAGACCCA-3′; reverse, 5′-TCTCGGCTGCTGCAGTTGG-3′ (305 bp); for β-actin, forward, 5′-AAATCGTGCGTGACATCAAA-3′; reverse, 5′-AGAAGGAAGGCTGGAAAAGA-3′ (180 bp). Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling (TUNEL) Assay. TUNEL assays were conducted with ApopTag Plus Fluorescein In Situ Apoptosis Detection Kit (S7111) following the indicated protocol. Briefly, C2C12 cells were seeded into 6-well plates at a density of 500,000 cells/well. After 24 h of incubation, the cell confluence reached 70−80%. After treatment, both attached and floating cells were harvested, fixed in 1% paraformaldehyde, washed in PBS three times, incubated in equilibration buffer for 10 min, and incubated with terminal deoxynucleotidyl transferase for 60 min at 37 °C. Cells were washed with stop buffer for 10 min and then incubated with antidigoxigenin conjugate for 30 min at 25 °C. After washing in PBS, cells were mounted with DAPI and viewed and photographed on a microscope with a fluorescent attachment. Immunocytochemistry. Cells were seeded on 22 × 22 mm coverslips (Corning Life Sciences, Lowell, MA) treated with poly-Dlysine. After fixing with cold 4% paraformaldehyde in PBS for 15 min and membrane permeabilization with 0.25% Triton X-100 at room temperature for 10 min, cells were blocked with 1% BSA in PBS containing 0.5% Tween-20 for 30 min followed by 1 h of incubation with primary antibodies at 37 °C and 1 h of incubation with FITC- or Cy3-conjugated secondary antibodies at room temperature. Cells were rinsed and mounted on coverslips with mounting medium containing DAPI (Vector Laboratories, Burlingame, CA). Fluorescence images were taken using a Zeiss LSM 510 Meta microscope (Carl Zeiss MicroImaging, Jena, Germany) with a 40× objective lens. Cell Transfection and Luciferase Activity Assay. C2C12 cells were seeded in 24-well plates. After overnight culture, the cell confluence reached about 80−90%. Next, 0.6 μg of p21-luciferase construct mixed with Lipofectamine 2000 at a ratio of 1:3 (w:v) in 100 μL of Opti-MEM I Reduced-Serum Medium was used for transfecting cells in one well. A pGL3 empty vector was used as a control. A pGL3 control vector containing the SV40 promoter and enhancer sequences was used to monitor the transfection efficiency simultaneously. Six hours after transfection, cells were moved to fresh medium, and 24 h after transfection, cells were exposed to MWCNTs. After the indicated time, cell lysates were prepared by adding 80 μL of 1× RIPA buffer to each well. The protein concentration was detected with a BCA Protein Assay Kit (Thermo Scientific, Waltham, MA). A luciferase activity assay was conducted in a white 384-well plate and detected with an Envision 2012 Multilabel Reader (PerkinElmer, Waltham, MA). Finally, 4 μL of the lysates and 25 μL of Steadylite Plus substrate (PerkinElmer) were used for a one-well assay. The luciferase activity was normalized as luminescence intensity divided by the protein concentration of the same lysate sample. Statistical Analysis. The two-sided Student’s t-test was used to analyze differences between experiments. Data are reported as the mean values ± SD from multiple determinations. P values of less than 0.05 were considered statistically significant, and all statistical calculations were carried out using SigmaPlot 10.0 (Systat Software).

MATERIALS AND METHODS

Cell Culture and Reagents. Carboxylated MWCNTs were prepared as previously reported.26 A stock solution of MWCNTs (1 mg/mL) in ddH2O was prepared and sterilized at 121 °C for 30 min and sonicated for 5 min at room temperature in an ultrasonic bath before use. Hek293 (human embryonic kidney epithelial cells) and C2C12 (mouse mesenchymal stem cells) were obtained from American Type Culture Collection (Manassas, VA) and were grown in Dulbecco’s minimum essential medium (DMEM; Gibco, Grand Island, NY). NB1691 (human neuroblastoma cells) obtained from the Pediatric Oncology Group cell bank27 were grown in RPMI 1640 (BioWhitaker, Walkersville, MD). All media were supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, Carlsbad, CA), 2 mM L-glutamine, 100 μg/mL penicillin, and 100 U/mL streptomycin. HCT-116 (human colon carcinoma) wild-type and variant cells depleted of the TP53 gene due to biallelic knockout28 were a generous gift from Dr. Michael Kastan (St. Jude Children’s Research Hospital).29 Mouse embryonic fibroblasts (MEFs; p21-deficient and wild-type) were generous gifts from Tyler Jacks (Massachusetts Institute of Technology, Cambridge, MA). Both wild-type and mutant HCT-116 and MEF cells were grown in DMEM supplemented with 20% heat-inactivated fetal bovine serum. Cells were incubated at 37 °C in a humidified atmosphere of 5% CO2 and 95% air. RNeasy Mini Kit was purchased from Qiagen (Germantown, MD). RIPA Buffer (10×, #9806) from Cell Signaling (Boston, MA) supplied with a Halt protease inhibitor cocktail (Thermo Scientific, Franklin, MA) was used for protein extraction. Antibodies including p53 (HR231), p21 (H164), cdk4 (C-22), cdk2 (M2), cdk6 (C-21), cyclin D1 (H-295), normal mouse (sc-2025), and rabbit (sc-2027) control IgG for coimmunoprecipitation, and Protein A/G PLUS-Agarose Immunoprecipitation Reagent (sc-2003) were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Cyclin B1 antibody (554178) was purchased from BD Biosciences (Billerica, MA). Rb (D20) antibody came from Cell Signaling. Goat antimouse and goat antirabbit secondary antibodies (#926-32220 and #926-32221) used for Western blot analysis came from LI-COR Biosciences (Lincoln, NE). All fluorescent-labeled secondary antibodies used for immunocytochemistry were commercial from Jackson ImmunoResearch (West Grove, PA). Cycloheximide came from Sigma-Aldrich (St. Louis, MO). Lipofectamine 2000 Transfection Reagent and Opti-MEM I Reduced-Serum Medium came from Invitrogen (Carlsbad, CA). The luciferase assay system (E1500) was purchased from Promega (Madison, WI). Guava Cell Cycle Reagent (4500−0220) came from Guava Technologies. Cell Cycle Analysis. Cells were seeded into 6-well plates at a density of 500,000 cells/well. After 24 h of incubation, the cell confluence reached 70−80%. MWCNTs were added to the designated concentrations. After the designated time, cells were trypsinized, aspirated, and counted. Then cells were centrifuged, washed with PBS, and stained with Guava cell cycle reagent for 30 min at room temperature in darkness prior to flow cytometry analysis. Co-Immunoprecipitation. Cell lysates were extracted with 1× RIPA buffer, and 200 μg of lysate was used for each immunoprecipitation. Before precipitation with primary antibodies, the lysate was precleared by adding 1.0 μg of the appropriate normal mouse or rabbit IgG (corresponding to the host species of the primary antibody) together with 20 μL of resuspended Protein A/G Plus Agarose and incubated at 4 °C for 30 min. Each precipitation used 1 μg of primary antibodies and 20 μL of Protein A/G Plus Agarose. After centrifugation at 1000g for 5 min, samples were boiled in 1% SDS solution and analyzed by Western blot. All Western blots were imaged with an Odyssey Infrared Imaging System (LI-COR Biosciences). mRNA Level Analysis. Cells were seeded in 6-well plates at a density of 500,000 cells/well. After 24 h of incubation, the cell confluence reached 70−80%. MWCNTs were added to the designated concentrations. After 24 h, cells were harvested for total RNA extraction. Reverse transcription polymerase chain reaction (RT-PCR) analysis was performed using 5 μg of total RNA extracted from the indicated cells. PCR amplification was carried out in 25 cycles, each

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RESULTS AND DISCUSSION Preparation and Characterization of MWCNTs. To make MWCNTs highly miscible in aqueous solutions, MWCNTs were partially oxidized to carboxylated MWCNTs. The resulting MWCNTs were purified by five rounds of washing and centrifugation after oxidation. This extensive purification procedure helped remove many impurities originally in the pristine MWCNTs, such as trace amount of heavy metals. The purity and physicochemical properties of MWCNTs30,31 were characterized and summarized in Table 1. Briefly, MWCNTs have an inner diameter of 5−12 nm and an outer diameter of 30−50 nm, and the lengths range from 0.5 to 1213



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adsorbed proteins. Inductively coupled plasma mass spectrometric analysis of 23 metals revealed that only a negligible amount of these metals was detected, excluding the possibility of unwanted effects from metal impurities.32,33 MWCNTs Retard Cell Proliferation by Cell Cycle Arrest. To gain a general understanding of the effects of MWCNTs on cell cycle progression, we used three different cell lines originating from humans and mice. Hek 293 is an embryonic human kidney cell line and a commonly used cell model for toxicological evaluations. Because MWCNTs hold promise in stem cell therapy such as in muscle and nerve tissue engineering,34,35 we also selected a mouse mesenchymal progenitor and myoblast cell line C2C12 and a human neuroblastoma cell line NB1691. MWCNTs bound to the cell surface (Figure S1A, Supporting Information) or were endocytosed into cells with continued membrane interactions (Figure S1B, Supporting Information). We found that MWCNT treatment at 25 and 100 μg/mL caused no obvious cell death in three cell lines, but the proliferation of cells was retarded in a dose dependent manner (Figure 1A−C). Decelerated proliferation without evident cell death suggested that a possibly hindered cell cycle progression was induced by MWCNTs. Flow cytometry results showed that all three cell lines were arrested at the G1/S transition. In addition, C2C12 and NB1691 cells were also slightly arrested at the G2/M transition 48 h after MWCNT treatment at a concentration of 100 μg/mL (Figure 1D). To further confirm this finding, we removed MWCNTs from the cell culture and washed cells with PBS after 24 h of incubation with nanotubes. The cells reentered cell cycling, and cell cycle arrest was diminished (Figure 1E). These results demonstrated that MWCNTs retard cell proliferation by cell cycle arrest. MWCNTs Cause p21 Accumulation and Cell Cycle Arrest. Cell cycle progression is mainly controlled by three

Table 1. Characterization of MWCNTs

2.0 μm (average of 1.0 μm). High-resolution transmission electron microscopy (HRTEM) images showed layers of nanotube walls. On the basis of the functional group loading (0.4 mmol/g) as determined by elemental analysis and the surface area of 500 m2/g (by manufacturer), we estimated that the average density of modification is approximately 5 functional groups per 10 nm2. At pH 7.0, MWCNTs carry negative charges. Their solubility in water and plasma is much better than that of pristine MWCNTs. Plasma protein bindings to MWCNTs shifted their zeta potential from −42.5 to −9.3 mV, indicating the neutralization of negative surface charges by

Figure 1. Cell cycle arrest in three cell lines treated with MWCNTs. (A−C) C2C12 cell proliferation observed by light microscopy after incubation under different concentrations of MWCNTs for 48 h. Scale bar: 200 μm. (D) The ratio of cell population at various cell cycle phases with MWCNT (100 μg/mL) treatment and those treated with medium. (E) After treatment for 24 h, MWCNTs were removed, and the cells were washed with PBS. The cells were incubated with fresh culture medium for another 17 and 36 h. Compared with the unwashed group, cells with removed MWCNTs re-entered the cell cycle as shown by the diminished cell cycle arrest indicated by the G0/1/S ratio. Results are expressed as the mean ± SE (n = 3) (*p < 0.05). 1214



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Figure 2. p21 mediated MWCNT-induced cell cycle arrest. (A) A schematic showing interactions of the cell cycle regulatory proteins in the regulation of cell cycle progression. The question mark indicates that the mechanism for p21 in inactivating the cyclin B/cdc2 complex is unclear. (B) Production of cell cycle regulatory proteins was analyzed by Western blot after cells were treated with MWCNTs (100 μg/mL) for 24 h. p21 expression was markedly increased. (C) Time-dependent cell cycle distribution in wild-type (wt) and p21 mutant (p21−/−) mouse embryonic fibroblasts (MEFs) measured by flow cytometry. Treatment with MWCNTs (100 μg/mL) arrested the cell cycle at the G0/G1-to-S transition as shown by the decreasing S phase cell ratio and increasing G2/M ratio as time progressed. No cell cycle arrest was found in the p21−/− cells. Results are expressed as the mean ± SE (n = 3). (D) C2C12 cells were treated by MWCNTs for 24 h, and cell lysates were immunoprecipitated with p21 antibodies followed by immunoblotting with various antibodies. p21/cdk4,6 and p21/cyclin D1 complex formation was enhanced by MWCNT treatment. (E) MWCNT (100 μg/mL) treatment for 24 h led to accumulation of hypophosphorylated Rb. N, without MWCNTs treatment; T, with MWCNTs treatment.

families of proteins known as cyclins, cyclin-dependent kinases (CDKs), and CDK-specific inhibitors (CDKIs).36 The former two classes of proteins form binary complexes, which contribute to the phosphorylation of retinoblastoma (Rb) protein, releasing the suppression of E2F protein and activating the expression of genes required for S phase entry.37 The CDKIs include the Ink4 family (p15, p16, p18, and p19) and the KIP/CIP family (p21, p27, and p57).38 KIP/CIP family of CDKIs block the kinase activity of CDKs by forming a ternary complex with the cyclin/CDK binary complex. Such complexes inhibit the phosphorylation of Rb and arrest the cell cycle at the G1 phase39 (Figure 2A). Alteration of the expression pattern of these proteins may lead to an aberrant cell cycle. In order to find out how MWCNTs caused cell cycle arrest, we first analyzed the expression levels of key members of the three families of cell cycle regulators to investigate whether they were altered by MWCNTs. We found that there was no change

in the levels of cyclin B1, cyclin D1, cdc2, cdk4, and cdk6. However, a 2.5- to 6.0-fold increase in p21 accumulation was observed in all three cell lines (Figure 2B). Protein p21 is one of the KIP/CIP family of CDKIs. Protein p21 forms a ternary complex with cyclin D/CDK4/6 or other cyclin/CDK complexes to regulate normal cell cycling. An alteration in p21 level may alter its interactions with cyclin D/CDK4/6 or other cyclin/CDK complexes37,39 and may arrest the cell cycle at the G1 phase.40,41 The MWCNT-induced accumulation of p21 prompted us to explore whether it is involved in cell cycle arrest. The gene knockout cell provides a useful model to study the functions of a particular protein.42 We used the wild-type and p21-knockout mouse embryonic fibroblasts (MEF) cells (MEF p21−/−) to analyze the role of p21 in MWCNT-induced cell cycle alterations. MWCNTs caused cell cycle arrest at the G1/S phase in wild-type cells. However, in MEF p21−/− cells 1215



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Figure 3. p21 was induced at the transcriptional level. (A) The stability of p21 in Hek 293 cells treated with MWCNTs (100 μg/mL) for 41 h or in medium was monitored by Western blot after the addition of the protein synthesis inhibitor cycloheximide (CHX, 100 μg/mL) for the indicated times. Protein expression level was quantified by the band density using the Image J method, and the ratio of p21 to β-actin bands at time 0 was normalized as 100%. (B) The p21 mRNA level was analyzed by semiquantitative RT-PCR analysis after MWCNT (100 μg/mL) treatment for 24 h.

Figure 4. MWCNTs did not induce cell apoptosis and activate p53 expression in C2C12 cells. (A) C2C12 cells in medium in 12-well plates were treated with MWCNTs for 24 h. Six hours before harvest, 2 μM doxorubicin was added as a positive control. Apoptosis was analyzed by the TUNEL assay. Apoptotic cell nuclei were stained by FITC (green) and observed under fluorescence microscopy. Scale bar: 200 μm. (B) Quantitative analysis showed that MWCNT treatment did not cause apoptosis in C2C12 cells. Average values and standard deviations were obtained from three independent experiments (ns, not statistically significant). (C) p53 expression level in three cell lines in untreated (N) and MWCNT-treated (100 μg/mL) (T) groups (24 h). p53 expression in C2C12 after treatment with 0.5 μM doxorubicin for 24 h was shown as a positive control.

MWCNTs induced no G1/S arrest (Figure 2C). These results suggested that an alteration of p21 protein might be required for MWCNT-induced cell cycle arrest. Then, the next question is how the accumulated p21 induces cell cycle arrest. In cells,

the phosphorylation of Rb protein is initially performed by the complex of cyclin D with cdk4 or cdk6 in late G1 phase,43 and this phosphorylation process is inhibited by the binding of p21 to the cyclin D/cdk4,6 complex.44 To test whether the elevated 1216



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Figure 5. MWCNT-induced expression of p21 was p53-independent. (A) MWCNTs (100 μg/mL) stimulated the expression of p21 but not p53 in wild-type (wt) and p53 mutant (p53−/−) HCT-116 cells. MWCNTs also inhibited the phosphorylation of retinoblastoma (Rb). The hypophosphorylated form of Rb (the form found in resting cells) increased upon addition of MWCNTs, and the hyperphosphorylated form (pRb, the form found in proliferating cells) decreased. (B) Immunocytochemical analysis of p53 and p21 responses to MWCNT treatment in wt and p53−/− HCT-116 cells. p53 was labeled with TRITC (red), and p21 was labeled with FITC (green). Cell nuclei were stained with DAPI (blue). Scale bar: 100 μm. (C) MWCNTs (100 μg/mL) arrested the cell cycle in both wt and p53−/− HCT-116 cells in a similar manner as measured by flow cytometry. The increasing ratio in G0/G1 phase and decreasing ratio in S phase indicated a G1/S arrest caused by MWCNT treatment. MWCNT treatment also induced G2/M arrest in both cell lines as shown by the high cell ratio in the G2/M phase. Results are expressed as the mean ± SE (n = 3).

inhibited protein biosynthesis with an inhibitor cycloheximide (CHX) and then analyzed the degradation rate of p21 with or without MWCNT treatment. Our experimental results showed that the rates of p21 degradation were comparable with or without MWCNT treatment (Figure 3A), indicating that MWCNTs did not affect the degradation rate of p21. Therefore, the MWCNT-induced increase in p21 protein accumulation (Figure 2B) should be a direct result of an enhanced p21 expression. To further confirm this, we analyzed p21 mRNA levels by polymerase chain reaction (PCR) analysis. The results indicated that MWCNT treatment enhanced transcription of the p21 gene in three cell lines (Figure 3B). Therefore, MWCNTs enhanced p21 protein overproduction at the mRNA level. DNA damage in cells stimulates p53 protein expression, which transactivates families of downstream genes and causes cell cycle arrest to provide extra time to repair damaged DNA.47 Protein p21 is a downstream target of p53.48 We wondered whether this p53 pathway is involved in MWCNT-induced p21 expression. First, we examined whether MWCNTs induced cellular DNA damage and apoptosis. By the terminal deoxynucleotidyl transferase dUTP nick end labeling

p21 level can actually inhibit the function of cyclin D/cdk4,6 complex, we carried out an immunoprecipitation experiment. We found that MWCNTs elevated the amount of p21/cyclin D1/cdk4,6 complexes (Figure 2D) and the hypophosphorylated Rb proteins in C2C12 cells (Figure 2E). By studying protein levels, protein complex, and p21 knockout cells, we found that MWCNTs caused an accumulation of p21 in cells and an increase in p21/cyclin D1/cdk4,6 complex formation, as well as an decrease in the phosphorylation of Rb protein. These data showed that the MWCNTs induced an overexpression of p21, which then inhibited Rb phosphorylation and caused cell cycle arrest at the G1/S phase. When the p21 expression machinery was removed in the p21 knockout cells, MWCNTs generated no detectable effects, further corroborating our findings. MWCNTs Induce Expression of p21 Not p53. Under the inhibition of protein biosynthesis, cellular proteins undergo autodegradation naturally. Either suppressed protein degradation or an enhanced protein expression may cause the accumulation of a protein in cells.45 Nanoparticles are known to alter protein stability toward degradation.46 To find out whether MWCNT treatment altered p21 degradation, we 1217



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Figure 6. p21 overexpression induced by MWCNTs was E-box-dependent. (A) Schematic representation of the p21 promoter fusions to luciferase used in this study, showing wild-type and mutant E-box sequences. The mutated bases are highlighted in red. (B) Transcriptional activation of the p21 promoter constructs by MWCNT (100 μg/mL) treatment for 48 h. The luciferase intensity in medium was normalized to 1.0. Average values and standard deviations were obtained from three independent transfection experiments (*p < 0.05).

Figure 7. Model showing a nonapoptosis-involved mechanism for arresting the cell cycle by MWCNTs. MWCNTs disturb BMP receptor functions and cause consequent inhibition of Id proteins, thus stimulating p21 expression by activating E-box motifs in the p21 promoter. The overexpression of p21 mediates cell cycle arrest in the G1/S or G2/M transition depending on the cell types. MWCNTs do not interact with the p53 binding domains in the p21 promoter region.

In both wild-type and p53 knockout (p53−/−) HCT-116 cells,28 MWCNT treatment induced comparable levels of p21 expression as shown by Western blot (Figure 5A) and immunocytochemistry (Figure 5B) without affecting p53 expression (Figure 5A). MWCNTs also caused the accumulation of hypophosphorylated Rb in both cell types (Figure 5A). These findings showed that neither MWCNT-induced p21 overexpression nor the inhibition of Rb phosphorylation required the p53 protein. MWCNTs also caused similar cell cycle arrest in both cell lines (Figure 5C), showing that p53 was not needed for MWCNT-induced cell cycle arrest. These findings substantiated our finding that MWCNT-induced cell cycle arrest was p53-independent. The perturbation of intrinsic cell signaling pathways can lead to cell cycle dysregulation. For example, we previously reported that carboxylated SWCNTs paused the cell cycle at the G1/S phase by perturbing the BMP signaling pathway without causing apoptosis.53 A similar perturbation on mitogenactivated protein kinase (MAPK) signaling pathway was also reported.25 BMP signaling pathway regulates a broad spectrum of functions involving cell proliferation and differentiation in embryonic and adult tissues.54 Therefore, we next investigated whether MWCNT-induced perturbation on BMP signaling and its downstream targets plays a role in cell cycle arrest. Induction of p21 Is E-Box-Dependent and under the Control of the BMP Signaling Pathway. The promoter region of the p21 gene includes binding sites for transcription

(TUNEL) assay, we found no detectable DNA damage in cells (Figure 4A) and no apoptosis (Figure 4B) after treatment at a concentration up to 100 μg/mL. CNT-induced cell apoptosis and DNA damage were occasionally reported. We realize that impurities in CNT might be responsible for such effects.49−51 We used a two-step oxidation method with five rounds of purifications after the synthesis of MWCNTs.26 The MWCNTs used in this study were repeatedly purified, as shown by elemental analysis of 23 metals.30 No apoptosis induction or DNA damages were expected with these MWCNTs since several previous studies have demonstrated that highly purified MWCNTs were less prone to inducing apoptosis.49−51 The lack of DNA damage implies that p53 expression may not be induced in cells. In order to confirm this, we analyzed p53 expression by Western blot. We found that there was no increase in the expression of p53 after MWCNT treatment in all three cell lines (Figure 4C), indicating that p53 expression was not altered by MWCNTs. These findings also suggested that p53 was probably not responsible for the overexpression of p21. This was further investigated next. Induction of p21 Protein Overexpression by MWCNTs Is p53-Independent. During development, p21 can be expressed in various tissues in a p53-dependent or p53independently manner.52 To firmly establish whether the MWCNT-induced p21 overexpression and cell cycle arrest were p53 independent, we investigated a p53 knockout cell line. 1218



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factors other than p53. One of these sites is the E-box motif.55 The E-box is a conserved DNA sequence to which a family of transcription factors containing basic helix−loop−helix (HLH) structures bind. Such bindings activate the expression of their controlled genes such as p21. Id proteins are also HLH proteins lacking DNA binding domains, and they are a downstream target of the BMP signaling pathway.56 CNTs downregulate the BMP signaling pathway and inhibit the expression of Id proteins in cells.53 We therefore speculated that MWCNTs might induce overexpression of p21 by an altered E-box regulation by Id proteins as a result of BMP signaling suppression. To verify this, we transfected C2C12 cells with a construct containing the luciferase gene driven by the p21 promoter.57 In this promoter, there are two E-box motifs (Figure 6A). Analysis of transcriptional activation of the p21 promoter showed that MWCNTs enhanced p21 promoter activity by about 3-fold and that this enhanced activation disappeared when E-box motifs in the promoter were dulled by mutations (Figure 6B). This showed that MWCNTs induced p21 overexpression via E-box modulation. The discovery of the critical involvement of the E-box in this mechanism provides a missing link between the findings from this investigation and our earlier findings that MWCNTs perturb the BMP receptor and the BMP signaling pathway. We have found that MWCNTs bind to BMP receptor 2 and inhibit the phosphorylation of BMP receptor 1 (manuscript submitted). This consequently leads to an attenuated BMP signaling transduction with a decreased phosphorylation of Smad proteins and downregulation of Id protein expression. Several genes including p21 are upregulated as Id proteins are downregulated. Combining our early findings (ref 53 and a submitted manuscript) and results from this investigation, we propose a working model to explain how MWCNTs affect the cell cycle (Figure 7). In this model, MWCNTs suppress the BMP signaling pathway and downregulate the production of Id proteins. The decreased Id protein level leads to the activation of E-box motifs in the p21 promoter region and cause p53independent overexpression of p21. Overexpressed p21 protein perturbs the cyclin D/cdk4,6 complex, reduces the phosphorylation of Rb, and causes cell cycle arrest.

AUTHOR INFORMATION

Corresponding Author

*Fax: 86-531-88380029. E-mail: [email protected]. Funding

This work was supported by the National Basic Research Program of China (2010CB933504), National Natural Science Foundation of China (90913006, 21077068, and 21137002), National Cancer Institute (P30CA027165), and the American Lebanese Syrian Associated Charities (ALSAC). Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Sharon Frase for technical assistance, Carme Gallego (Universitat de Lleida) for providing the p21-luciferase construct, and Michael Kastan and Tyler Jacks (MIT, Cambridge) for providing wt/p53−/− HCT-116 cell lines and wt/p21−/− MEF cell lines.

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ABBREVIATIONS MWCNTs, multiwalled carbon nanotubes; BMP, bone morphogenetic proteins; Id proteins, inhibitor of DNAbinding/differentiation proteins; CDK, cyclin-dependent kinase; CDKI, CDK-specific inhibitor; Rb, retinoblastoma protein; ROS, reactive oxygen species; MEF, mouse embryonic fibroblasts; PCR, polymerase chain reaction; TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling; HLH, helix−loop−helix; CHX, cycloheximide; FITC, fluorescein isothiocyanate

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REFERENCES

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CONCLUSIONS Our investigations reveal a series of molecular events and demonstrate how carboxylated MWCNTs induce p53independent p21 expression that caused cell cycle arrest. We previously found that MWCNTs bound to BMP receptor 2 and suppressed BMP signaling, which led to the downregulation of Id proteins. The p21 overexpression is a result of the regulation of the E-box motifs in the p21 promoter by the declined concentration of Id proteins. The overexpressed p21 protein then binds to the cyclin D/cdk4,6 complexes and inhibits Rb phosphorylation. Hypophosphorylation of Rb prevents the release of E2F factors and causes cell cycle arrest. Findings from this research provide valuable insight into a mechanistic understanding of CNTs’ effects on cellular functions and will make strong impacts on nanomedicine and nanotoxicity research.

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S Supporting Information *

TEM images of MWCNTs binding to cell surface or being endocytosed into cells. This material is available free of charge via the Internet at http://pubs.acs.org. 1219



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Cell Cycle Regulation by Carboxylated Multiwalled Carbon

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